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J. Nat. Prod. 2005, 68, 280-281
Mycgranol, a New Diterpene from the Marine Sponge Mycale aff. graveleyi Amira Rudi, Yehuda Benayahu, and Yoel Kashman* School of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel Received July 26, 2004
Mycgranol, a new isocopalane diterpene (methyl 1β-hydroxy-2-oxoisocopalanoate) was isolated from the Kenyan sponge Mycale (Aegogropila) aff. graveleyi. The structure of the compound was elucidated by interpretation of MS, COSY, HMQC, HMBC, and ROESY data. The absolute configuration of mycgranol was determined to be the same as that of the nudibranch isocopalane diterpenes rather than that of the ent-compounds known from sponges of the genus Spongia. As part of our ongoing efforts to isolate biologically active compounds from marine invertebrates,1,2 the constituents of the Kenyan long-digitated tubular orange sponge Mycale (Aegogropila) aff. graveleyi, Burton (Demospongiae, Poeciloscleridae, Mycalidae), collected in the Shimoni Channel, Kenya, were examined and a new diterpene designated as mycgranol (1) was isolated. Although more than 20 subspecies of Mycale were investigated resulting in many unique secondary metabolites,3-5 M. graveleyi has thus far not been explored and no tricyclic diterpene was reported. Diterpenes are well known from marine invertebrates, and a large variety of skeletons have been reported.3-5 Among those, ent-isocopalane diterpenoids are widely present in marine sponges;6,7 for example, from the Mediterranean Spongia zimocca were isolated several oxygenated ent-isocopalanes such as 15-hydroxy-ent-isocopal-12en-16-al.7 The enantiomeric isocopalane diterpenoids were mainly isolated from nudibranches as part of acylglycerols.8 The carbon skeleton of these diterpenoids is rather rare in nature. The ethyl acetate/CH3OH (1:1) extract of the freezedried sponge was subjected to solvent partition,9 and the petroleum ether fraction was chromatographed on Sephadex LH-20 and silica gel columns to afford mycgranol (1) (40 mg, 0.8% dry weight). The EI mass spectrum of mycgranol (1) exhibited a molecular ion M+ at m/z 348. The 13C NMR, 1H NMR, and 2D spectra (Table 1) revealed the presence of the following moieties: (a) a ketone (δC 212.0), (b) an external double bond (δC 143.1 C, 108.6 CH2), (c) a methyl ester (δC 171.8 C, 50.9 CH3), and (d) a hydroxyl group (δC 79.1 CH). On the basis of this analysis, the molecular formula was determined to be C21H32O4, and to fulfill the six degrees of unsaturation, 1 has to be tricyclic. First, the strong HMBC correlations of the four methyl groups and the methylene with their neighboring C atoms were analyzed, and the resulting units were then extended by COSY correlations (Figure 1). Correlations in the HMBC spectrum from H-1 and H-3 to the ketone carbonyl group and from the OCH3 (H-21) and H-14 to the ester CO2 group established the position of the carbonyl (C-2) and the methoxycarbonyl (C-15) groups. The downfield resonances of the AB system of H2-3 (2.85 and 2.03 d) and the singlet of H-14 (2.89 s) confirmed the position of the carbonyl group. Additional HMBC correlations (Table 1) corroborated the suggested planar structure of 1. The relative stereochemistry of mycgranol was determined from the coupling constants and correlations ob* To whom correspondence should be addressed: Tel: +972-3-6408419. Fax: +972-3-6409293. E-mail:
[email protected].
10.1021/np0497550 CCC: $30.25
Table 1. NMR Data for Mycgranol (1) in CDCl3 (500 MHz for 1H and 100 MHz for 13C)a C# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
δC
δH (m, J in Hz)
79.1 CH 3.58 s 212.0 C 50.8 CH2 2.85 d (12.8) 2.03 d (12.8) 39.0 C 47.9 CH 1.75 dd (13.9, 2.0) 18.8 CH2 1.60 m 1.45 m 39.3 CH2 1.63 m 1.32 m 39.6 C 48.7 CH 1.89 dd (12.0, 2.8) 45.0 C 22.2 CH2 1.48 m 1.42 m 35.6 CH2 2.40 ddd (12.8, 4.1, 2.0) 2.10 dt (12.8, 2.0) 143.1 C 63.1 CH 2.89 s 171.8 C 108.6 CH2 4.82 s, 4.66 s 14.9 CH3 1.07 s 23.2 CH3 0.87 s 32.9 CH3 1.03 s 16.1 CH3 0.86 s 50.9 CH3 3.62 s
HMBC (C to H)b 9, 20 1, 3a, 3b 18, 19 3a, 3b, 5, 18, 19 3a, 3b, 7a, 18, 19, 20 5, 7a, 7b 5, 6a, 9, 17 9, 14, 17 14, 17, 20 5, 20 9 14, 16a, 16b 12a, 12b, 14, 16a, 16b 9, 12a, 16a, 16b, 17 14, 21 12a, 12b, 14 7a, 7b, 14 3a, 3b, 5, 19 3a, 3b, 5, 18 1, 9
a Multiplicities of the carbon signals were determined by DEPT, and all assignments were confirmed from 2D experiments. b a and b denote downfield and upfield resonances within a geminal pair.
Figure 1. COSY (s) and key HMBC (∩) correlations for 1.
Figure 2. Key NOEs for 1.
served in the ROESY spectrum (Figure 2). Thus, H-5 and H-9 have to be both axial according to large Jax,ax values, with H-6ax and with H-11ax, respectively (Table 1).
© 2005 American Chemical Society and American Society of Pharmacognosy Published on Web 02/08/2005
Notes
Figure 3. Octants projection for 1.
ROESY correlations between methyls 17 and 20 and between 18 and 20 established the R-axial position of all three. The proposed configuration also agrees with their relative upfield resonances (δC 15-23 ppm) due to γ-effects. The all-trans structure was confirmed from several NOEs on the β-side of 1 as shown in Figure 2. The absolute configuration of compound 1 was determined from its Cotton effect measured from the CD curve (∆ +0.25). The nfπ* excitation of the C-2 carbonyl group is expected to be mainly perturbed by the neighboring 1βaxial-hydroxyl group which is in the first sphere of the CO group.10,11 The axial OH group (against equatorial groups which are on the nodal plane and have little effect) causes a bathochromic shift to λmax 307 mµ and an increase of the Cotton effect. According to the octant rule, the hydroxyl group has to be in the rear lower-right octant as depicted in Figure 3. Ring C (Figure 3) with its substituents is in the fourth sphere10 and expected to have a weak negative contribution to the Cotton effect, if there is any. Hence, the 1S configuration was determined for 1, and accordingly, the absolute configuration of 1 is as in the anisodorinisocopalane diterpenes isolated from the mantle of the dorid nudibranch Anisodoris fontaini.8 The proposed configuration is enantiomeric to the ent-diterpenes isolated from sponges of the genus Spongia.7 Mycgranol (methyl 1βhydroxy-1-oxoisocopalanoate) is the first example of an isocopalane-diterpene from a sponge. Experimental Section General Experimental Procedures. Optical rotations were obtained with a Jasco P-1010 polarimeter. Circular dichroism measurements were performed on an AVIV 202 spectrometer. IR spectra were obtained with a Bruker FTIR Vector 22 spectrometer. 1H and 13C NMR spectra were recorded on Bruker ARX-500 and Avance-400 spectrometers. 1 H, 13C, COSY, HMQC, and HMBC were recorded using standard Bruker pulse sequences. EIMS and HREIMS measurements were recorded on a Fisons, Autospec Q instrument.
Journal of Natural Products, 2005, Vol. 68, No. 2 281
Biological Material. Mycale (Aegogropila) aff.graveleyi, Burton, a long-digitated tubular orange sponge, was collected in the Shimoni Channel off Mombasa, Kenya, by scuba at a depth of 8-11 m in February 2003. A voucher sample, ZMAPOR 17808, is deposited at the Zoological Museum, Tel Aviv University. Extraction and Isolation. Freeze-dried sponge (5 g) was homogenized and successively extracted with ethyl acetate/ CH3OH (1:1). The extract (900 mg) was subjected to partition by the method of Kupchan et al.9 to afford five fractions (petroleum ether, CCl4, CHCl3, n-BuOH, and water). The petroleum ether fraction (200 mg), containing 1 with sterols and glycerides, was repeatedly chromatographed on a Sephadex LH-20 column, eluting with a mixture of hexane/CHCl3/ CH3OH (2:1:1), followed by a silica gel column [hexane/ethyl acetate (7:3)] to afford mycgranol (1) (40 mg, 0.8% dry weight). Compound 1: colorless oil; [R]D +2.5 (c 2.5, CHCl3); IR (CHCl3) νmax 2950, 1735, 1717, 1649 cm-1; 1H and 13C NMR data, see Table 1; CIMS m/z (%) 348 (100), 331 (21), 317 (37), 299 (18), 289 (11); HREIMS obsd [M+] m/z 348.2305 (calcd for C21H32O4, 348.2301). Acknowledgment. We thank PharmaMar Madrid for financial support and R. Van Soest, Amsterdam, for the identification of the sponge. Special thanks are due to C. O. Oduol, Assistant Director of Fisheries, Mombasa, Ministry of Agriculture and Rural Development, Fisheries Department, Kenya, for issuing the collection permit for the research expedition. References and Notes (1) Chill, L.; Rudi, A.; Benayahu, Y.; Schleyer, M.; Kashman, Y. Org. Lett. 2004, 6, 755-758. (2) Rudi, A.; Shalom, H.; Schleyer, M.; Benayahu, Y.; Kashman, Y. J. Nat. Prod. 2004, 67, 106-109. (3) Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1-48, and previous reports in this series. (4) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2003, 20, 1-48; 2004, 21, 1-49. (5) Blunt, J. W.; Munro, M. H. G. “marinlit” Database 2004, University of Canterbury, Christchurch, New Zealand. (6) de Miranda, D. S.; Brendolan, G.; Imamura, P. M.; Sierra, M. G.; Marsaioli, A. J.; Ruveda, E. A. J. Org. Chem. 1981, 46, 4851-4856. (7) Zubia, E.; Gavagnin, M.; Scognamiglio, G.; Cimino, G. J. Nat. Prod. 1994, 57, 725-731. (8) Gavagnin, M.; Ungur, N.; castelluccio, F.; Muniain, C.; Cimino. G. J. Nat. Prod. 1999, 62, 269-274. (9) Kupchan, S. M.; Komoda, Y.; Branfman, A. R.; Sneden, A. T.; Court, W. A.; Thomas, G. J.; Hintz, H. P. J.; Smith, R. M.; Karim, A.; Howie, G. A.; Verma, A. K.; Nagao, Y.; Dailey, R. G., Jr.; Zimmerly, V. A.; Summer, W. C., Jr. J. Org. Chem. 1977, 42, 2349-2357. (10) Snatzke, G., Ed. Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry; Heyden and Son Ltd.: London, 1967. (11) Eliel, L. E.; Wilen, S. H.; Mander L. N. Stereochemistry of Organic Compounds; John Wiley and Sons Inc.: New York, 1994; p 991.
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